Aftins increase amyloid-β42, lower amyloid-β38, and do not alter amyloid-β40 extracellular production in vitro: toward a chemical model of Alzheimer's disease?

Abstract

Increased production of amyloid-β (Aβ)42 peptide, derived from the amyloid-β protein precursor, and its subsequent aggregation into oligomers and plaques constitutes a hallmark of Alzheimer's disease (AD). We here report on a family of low molecular weight molecules, the Aftins (Amyloid-β Forty-Two Inducers), which, in cultured cells, dramatically affect the production of extracellular/secreted amyloid peptides. Aftins trigger β-secretase inhibitor and γ-secretase inhibitors (GSIs) sensitive, robust upregulation of Aβ42, and parallel down-regulation of Aβ38, while Aβ40 levels remain stable. In contrast, intracellular levels of these amyloids appear to remain stable. In terms of their effects on Aβ38/Aβ40/Aβ42 relative abundance, Aftins act opposite to γ-secretase modulators (GSMs). Aβ42 upregulation induced by Aftin-5 is unlikely to originate from reduced proteolytic degradation or diminished autophagy. Aftin-5 has little effects on mitochondrial functional parameters (swelling, transmembrane potential loss, cytochrome c release, oxygen consumption) but reversibly alters the ultrastructure of mitochondria. Aftins thus alter the Aβ levels in a fashion similar to that described in the brain of AD patients. Aftins therefore constitute new pharmacological tools to investigate this essential aspect of AD, in cell cultures, allowing (1) the detection of inhibitors of Aftin induced action (potential 'anti-AD compounds', including GSIs and GSMs) but also (2) the identification, in the human chemical exposome, of compounds that, like Aftins, might trigger sustained Aβ42 production and Aβ38 down-regulation (potential 'pro-AD compounds').

Fig. 1

Structure of the compounds used in this study. N⁶-diamino-purine (N6DA) (1), compound (2), Aftin-1 (3), Aftin-2 (4), Aftin-3 (5), Aftin-4 (6), Aftin-5 (7), (R)-roscovitine (8) and “Torrey Pines” compound (9).

Fig. 2

Dose-dependent effect of various purines on extracellular Aβ₄₂ and Aβ₄₀ production in N2a-AβPP695 cells. Cells were exposed to various concentrations of compounds 1–6, 8 for 18 h. Extracellular Aβ₄₂ (upper panel) and Aβ₄₀ (lower panel) levels were measured by an ELISA assay and are expressed as fold change over the level of control, vehicle-treated cells. Representative of two independent experiments, errors bars represent standard deviation of triplicate values.

Fig. 3

Effect of Aftin-5 on extracellular Aβ production in N2a-AβPP695 cells. A) Aftin-5 dose-dependent effect on extracellular amyloids levels. Cells were exposed to various concentrations of Aftin-5 for 18 h. Extracellular Aβ₃₈, Aβ₄₀, and Aβ₄₂ levels were measured by an ELISA assay and are expressed as fold change over the level of control, vehicle-treated cells. B) Aftin-5 time-dependent effect on Aβ₄₀ and Aβ₄₂ levels. Cells were exposed to 100 μM Aftin-5 for various periods of time and extracellular Aβ₄₀ and Aβ₄₂ levels were measured by an ELISA assay. Representative of three independent experiments, errors bars represent standard deviation of triplicate values.

Fig. 4

Extracellular Aβ₄₂ production induced by Aftin-5 and basal level of Aβ₄₀ are inhibited by γ-secretase inhibitors. Cells were exposed to increasing concentrations of DAPT or BMS 299897 and 1 h later to 100 μM Aftin-5. Extracellular Aβ₄₀ and Aβ₄₂ levels were measured after 18 h and are expressed relative to their levels produced by Aftin-5 treated cells in the absence of γ-secretase inhibitor. Representative of two independent experiments, errors bars represent standard deviation of triplicate values.

Fig. 5

γ-Secretase modulator ‘Torrey Pines’ counter-balances Aftin-5 effects. A) GSM ‘Torrey Pines’ dose-dependent effect on extracellular Aβ₃₈, Aβ₄₀, and Aβ₄₂ levels in N2a-AβPP695 cells. Cells were exposed to various concentrations of ‘Torrey Pines’ compound for 18 h. Extracellular Aβ levels were measured by an ELISA assay and are expressed as fold change over the level of control, vehicle-treated cells. B) GSM ‘Torrey Pines’ dose-dependently inhibits Aftin-5 upregulation of Aβ₄₂ and down-regulation of Aβ₃₈. Cells were exposed to various concentrations of ‘Torrey Pines’ compound and 1 h later to 100 μM Aftin-5. Incubation was carried out for 18 h. Extracellular Aβ levels were measured by an ELISA assay and are expressed as fold change over the level of control, Aftin-5 treated cells. Representative of three independent experiments, errors bars represent standard deviation of triplicate values.

Fig. 6

Aftin-5 modifies extracellular but not intracellular Aβ₃₈/Aβ₄₀/Aβ₄₂ ratios. Cells were exposed to 100 μM Aftin-5 for 18 h, in the absence or presence of 1 μM DAPT or 1 μM ‘Torrey Pines’ compound. Cell supernatant (A) and cell pellets (B) were recovered and Aβ₃₈, Aβ₄₀, and Aβ₄₂ amyloid levels were measured by an ELISA assay. Amyloid levels are expressed as fold change over their concentrations in control, non-treated cells. Basal extracellular (A) and intracellular (B) levels of amyloids were, respectively, 61.9 and 83.5 pg/mL (Aβ₃₈), 471 and 48.9 pg/mL (Aβ₄₀), 28.9 and 33.6 pg/mL (Aβ₄₂). Representative of two independent experiments, errors bars represent standard deviation of triplicate values.

Fig. 7

Aftin-5 has modest effects on mitochondrial structure. A) Ultrastructure of isolated mitochondria exposed to 100 μM Aftin-5, 50 μM Calcium, or 50 μM Calcium + Cyclosporin A (CsA). B) Ultrastructure of mitochondria within cells exposed to 100 μM Aftin-5 for 6 or 24 h, or to 5 μM doxorubicin. Representative of two independent experiments.